1538.

SYNTHESIS, REDUCTION, AND PHARMACOLOGICAL PROPERTIESOF THE SULFOXIDES OF SOME CARRA-ANALOGUESOF OXYTOCIN*

Michal LEBL, Tomislav BARTH and Karel JOSTInstitute of Organic Chemistry and Biochemistry,Czechoslovak Academy of Sciences, 16610 Prague 6

Received July 26th, 1977

Direct selective oxidation afforded the sulfoxides of deaminc-Ivcarba-oxytocin (Ia), deamino­-ti-carba-oxytocin (Ie), and Lcarba-oxytocin (Id). The sulfoxide Ia was also prepared by a step­wise synthesis of the peptide chain. For purposes of comparison, the sulfone of deamino-l-carba­-oxytocin (Ib) was prepared. The pharmacological properties ofcompounds Ia-Idwere examined.Of a special interest is the difference in activities of sulfoxidee of the l-carba and ti-carba seriesand dissociation of effects on uterus and lactation. With the use of model compounds, the reduc­tion of suIfoxides was examined and the most advantageous procedure (hydrogen bromide/ace­tone) was applied to the sulfoxide Ia which afforded the sulfide Ie without formation of by-pro­ducts.

The sulfides are known to undergo oxidation more readily than the disulfldes".It may be thus expected that the so-called carba-analogues of neurohypophysial hor­mones will be more exposed to the danger of oxidation reactions than the naturallyoccurring disulfide hormones.** Since the structural requirements and influence of thestructure in the region of the disulfide bridge on the biological activity have notbeen so far examined except for the snbstitution of sulfur by the methylene group' - 5

or selenium", it is hardly possible to predict any biological properties of the oxidizedcarba-analogue. According to a hypothesis, the snlfoxide of deamino-l-carba-oxy­tocin conld exhibit a low activity' and its formation could canse the decreased activityof older preparations7, but this idea has been neither confirmed nor controverted.By the formation of a sulfoxide, there is explained the inactivation Or decreasedactivity of ACTH (ref. 8 •9) and its fragments!", MSH (ref. 11 ), calcitoninv', parat­hormone", eledoisin 14 or carboxyterminal tetrapeptide gastrin" when these sub­stances are treated with aqueous hydrogen peroxide. In this connection, some of thesehormones containing the snlfoxide of methionine have been synthesized12 ,! 4 . " .

'" Part Cl'l. in the series of Amino Acids and Peptides; Part CXLVIII: This Journal 43, 1300(1978).*'" The nomenclature and symbols obey the earlier published recommendations-. The aminoacids appearing in this paper are of the t-serles. The abbreviations Hey and Pier designatehomocystein and picric acid, resp.

Collection czecboelov. Chern. Commun. [Vol. 431 [19781

Sulfoxidcs of Carba-Analogues of Oxytocin 1539

The sulfide can be converted to the sulfoxide by several procedurea'P from which,however, only a few are suitable for the present field of substances. The procedureof choice must afford selectively the sulfoxide, the reaction conditions must be mild,and the reaction must be as fast as possible. Analogous requirements apply to reduc­tion of the sulfoxide.

As a model substance in investigations on the oxidation of sulfides and reductionof the corresponding sulfoxides, S-(y-methoxycarhonylpropyl)cysteine3 (IVa) wasused, i.e., the parent amino acid of the so-called l-carba-analogues of neurohypo­physeal hormones. Oxidation with hydrogen peroxide in acid and neutral mediawas examined. The sulfone VI a is quantitatively formed in acid media whereasa slow oxidation to the sulfoxide Va occurs in neutral media. Twenty four boursat room temperature are required for a complete conversion. When compared withcystathionine'? and the thialysine derivatives! B, the present reaction is more specific.In the case of cystathionine, the formation of the sulfoxide was observed' 9 even in acidmedia under vigorous conditions. A similar behaviour was also reported for thecase of methionine-? which affords a pure sulfoxide by the action of excess hydrogenperoxide inIM-HCI. When the oxidation is performed with performic acid, the resultsare analogous to those obtained in the case of Svmethylcysteinej' (II). At roomtemperature, the sulfone VIa is exclusively obtained whereas at -15°C, a mixtureof the snlfone VI a and the sulfoxide Va is formed. Like the German anthors' 8 workingon thialysine derivatives, we did not succeed in preparing a pure sulfoxide by theaction of bromiue. Best results were obtained with the use of the periodate as oxidanr'"(this reagent has not been as yet used in the oxidation of amino acids). The reactionis fast enough and the first order reaction coustantequals to 1·1 .10-3 • s-· at 22'C.The use of excess reageut and prolonged reaction time does not result in the formationof the sulfone. The conversion of the sulfide is quantitative after 2 h; after additional200 h, uo sulfone was detected by chromatography. The advantage of potassiumperiodate consists in its low solubility in water and the great solubility of the iodateas tbe reaction product (in spite of excess reagent, the reaction takes place in an excessof the oxidized component). Sodium periodate is much more soluble in water andits reduction product, sodium iodate, can be precipitated with methanol after com­pletion of the oxidation.

Interesting results may be observed in the acid hydrolysis (6M-HCl, 105'C underdiminished pressure) of the sulfoxide Va. Irrespective of the reaction time, the hydro­lysis of the sulfoxide (either free or as component of a peptide chain) affords S-(y-car­boxypropyl)cysteine (Vb) in 50% yield. A similar behaviour may be observed in thecase of S-(J3-melhoxycarbonylethyl)homocysteine sulfoxide (VIII) and cystathioninesulfoxide as components of the peptide. Such a behaviour was also reported in thecase of S-methylcysteine sulfoxidef! (III) but was not quantitatively evalnated.Under analogous conditions, methionine sulfoxide affords methionine in highyield 2 2 (cJ. ref,") whereas S-carboxymethylcysteine sulfoxide gives a mixture ofpro-

Collection Czechoslov. Chern. ccmmun. [Vol. 431 11978]

1540 Lebl, Barth, Jest :

ducts in which cysteine predominatesf". Contrary to S-carboxymethylcysteine sul­fone 21 , the sulfones VIa and IX and S-methylcysteine sulfone do not decompose(only the methyl ester is hydrolysed); we may thus exclude explanation of the 50%recovery of the sulfide from the sulfoxide Va by means of a disproportionation to thesulfide IV and the sulfone VI followed by decomposition of the sulfone with theformation of ninhydrin-negative substances. It was not possible to exclude a priorianother mechanism consisting in the decomposition of the sulfoxide Va to a sulfenicacid derivarlve'" (and dehydroalanine which is converted to pyruvic acid); the sul­fenic acid derivative would make possible reduction of the other portion of the sulfo­xide Va to the corresponding sulfide. However, in the case of the sulfoxide VIIIsuch an explanation based on a partial reduction is not as evident since the yieldof S-(p-carboxyethyl)homocysteine VIIb in the hydrolysis of the sulfoxide VIIIwas 20%in the case of the free amino acid and 54%in the case of the peptide Ic. By theformation of a snlfoxide and its snbseqnent 50% destruction it is possible to explainthe low values of S-(y-carboxypropyl)cysteine (IVb) in the amino-acid analysis andto determine in this manner the content of the sulfoxide V in the peptide. The hydro­lysis of the substance is performed in a properly evacuated (10 min) and sealedampoule since in the case of an incomplete evacuation or when the evacuationis omitted, the yield of the amino acid is as low as 70%. When the hydrolysis isperformed in the presence of an oxidant (NaIO4), the recovery of the sulfide I Vbis not recorded and the hydrolysate contains the original sulfoxide Vb (in the formof a diacid]; such a result does not correspond to the above proposed mechanism.

From circnlar dichroism curves of the present sulfoxides, the absolute configurationon sulfur of the predominant diastereoisomer was determined as R (ref.24) . In the

TABLE I

Rp and E Values of Some Amino Acids and Their Oxidation Products

Com- RF E G l)'pound Sl S5"

2.4S2 S3 S4

IVa 0'39 0'17 0'29 0'46 0'58 0'41Va 0'15 0·09 0'10 0'26 0'45 and O'34 0'30VIa 0'22 0'13 0'20 0'40 0'36 0'24VIla 0'35 0'12 0'17 0'26 0·65 0·61VIII 0'10 0'03 0'07 0'12 0'50 0·50IX 0'18 0·04 0'12 0'23 0'00-0,65 0'45

Q Paper chromatography.

---Collection Ozechcelcv. Chern.commun. [Vol. 43] [1976]

Sulfoxides of Carha-Analogues of Oxytocin 1541

periodate oxidation of S-(y-methoxycarbonylpropyl)cysteine (IVa), the ratio of theresulting diastereoisomers is 54 : 46; in the oxidation with hydrogen peroxide, theratio is 57 : 43. These diastereoisomeric sulfoxides may be separated by paper chro­matography and ion exchange chromatography under conditions of an analysisin amino-acid analyser, see Table I and II. The separation by ion exchange chro­matography is also successful in the case of diastereoisomeric sulfoxides VIII butfails with diastereoisomers of S-methylcysteinesulfoxide (III).

TABLE II

Elution Times of Some Amino Acid Derivatives in Amino-acid Analyzer

Compound Time, min Compound Timec min

IVa 117 II 72Va 46 and 48 III 43VIa 47-5 VIII 50 and 51'5IVb 93 IX 63

Vb 43 and 45 Asp 48

VIb 43 Gly 85VIla 86 lie 123VIlb 116 Phe 141

TABLE III

Chromatographical Data (R F) of Some Oxytocin Analogues

Collection caechostov. Chern. Commun. (Vol. 43] 11978]II

Com­pound

IaIbIcIdIeIf

[0

n Paper chromatography.

SI

0-160-190-130-020·220-180-03

S2

0-120-110-110-000-170'140-01

S3

0-230-260-020-000'210-030-00

S4

0-620-620-450-070-620-52

0'06

S5"

0-48

0-57

1542 Lebl, Barth, Jolt:

In the preparation of sulfoxides of carba-analogues of oxytocin, the sodium perio­date oxidation was used as the procedure of choice. After two hours, the excessoxidant and the corresponding iodate were removed by gel filtration and the productwas freeze-dried. This procedure was used in the preparation of sulfoxides I a, I c,and Id derived from deamino-J-carba-oxytocin, deamino-S-carba-oxytocin, andl-carba-oxytocin, resp. In order to demonstrate that no sulfone is formed from thesulfide in this case, the authentic sulfone Ib of deamino-l-carba-oxytocin was pre­pared by performic acid oxidation and its chromatographic data were examined.The chromatographic behaviour (Table HI) of the sulfone Ib was different from thatof the sulfoxide I a and the product of the periodate oxidation did not contain evena trace amount of the sulfone lb. When a solution of the sulfide Ie in methanolwas kept at room temperature for half a year, formation of the sulfoxide Ia wasdetected by chromatography.

~: CH,

/ ~~, /C1-I,

R H. CH

CH.-~H-CO-NH-~I'I':"'CO-NH-~H-CO/, -

7 .CH,-~H-NH-CO-CH-NH-<;:o-CH-NH

I 0 ~H,-CONH, ~H,-crl,-CONH,N

/ -,~H, ~H-Co-NH-TH-CO-NH-CH,-CONH,

H,- 'I, TH,

CH/"­

CH;; CH 3

l u. X ~ CH,-SO. R ~ Hlb. X ~ CH,-SO,. R = 'Iu, X ~ SO-CH,. R ~ HId. X ~ CH,-SO. R ~ NH 2

t« X ~ CH 2- S.If. X ~ S-C1i 2•

If!. X ~ CH,-S.lb. X ~ CH 2-C!:I,.

R=HR~H

R ~ NH,R=H

The amino acid analysis may be successfullyapplied to prove sulfoxide and sulfonestructures. Sulfoxide Ia gives 50% of parent amino acid in the form of sulfide IVb,whereas sulfone of S-(y-carboxypropyl)cysteine(Vlb) remained unchanged. Further-

Collection Czechoslov.Chern. commun. [Vol. 43] [19781

Sulfoxides of Carba-Analogues of Oxytocin 1543

more, the vibrations of -SO- and -S02- gronps can be detected by IR spectro­scopy. The spectrum of a model mixture containing 90% of deamino-dicarba-oxy­tocin (Ill) and 10% of sulfoxide 111 contains a band at 1006 cm- 1 of medium inten­sity, whereas the spectrum of compound 1a has two weak bands in the expectedarea with frequencies shifted to the greater values. This finding should indicate theintramolecular interaction of sulfoxides with some polar group of the peptide mole­cule. Such an interaction is demonstrated by splitting the band into a doublet withlower intensity and therefore it is not possible to use IR spectroscopy to detectsmall amounts of sulfoxides in the analogue synthesized.

Polarography seemed to be promising for detection of sulfoxides". S-Methyl­cysteine sulfoxide (III) and sulfoxide Va were used as model compound. In the firstcase no wave attributable to the reduction of sulfoxide was observed and in the othercase only a poorly developed wave was detected just before the area of the finalcurrent increase. This wave may be attributed to the catalytic wave of hydrogenand it is not therefore possible to Use it for the detection of sulfoxides in the moleculeswith molecular weights similar to those of neurohypophysial hormones. The Thomp­son tesr'", which is still the most specific assay, although not quantitative, was usedfor verifying the newly synthesized sulfoxides.

For completely unambiguous confirmation of the identity of the synthesized analo­gues the stepwise building up of deamino-f-carba-oxytocin sulfoxide (Ia) was per­formed. This analogue was selected as a representative of the sulfoxide series becauseof synthetic procedure reasons and a dissociation of biological activities accompaniedby a decrease of biological potencies. Sulfoxide Va was converted by means of Buntesalt2 7 to N-benzyloxycarbonyl-S-(y-methoxycarbonylpropyl)cysteine sulfoxide, whichwas obtained in the form of dicyclohexylammonium salt (X). The extremely lowyield of this reaction obviously resulted from the intramolecular interaction of theamino group with the sulfoxide that served as protection against acylation. Carbo­benzoxylation using benzyloxycarbonyl chloride at higher pH (8·5) gives the desiredproduct X in a good yield. Owing to the fact that partial reduction of sulfoxideof S-benzylcysteine was describedj" when the benzyloxycarbonyl group from thecysteine derivative was split off by hydrogen bromide in acetic acid, we verifiedwhether similar reduction did not occur also in the case of sulfoxide Va. By TLCwe found that approximately 5% of sulfide was regenerated and for this reason theprocedure was avoided.

Tetrapeptide amide Xib obtained by decarbobenzoxylation of peptide Xia (ref.")was oxidized with sodium periodate. Sulfoxide XII in contrast to peptide Xlb wasobtained in a crystalline form, in accordance with tbe expected better crystallisationproperties of sulfoxidesjP. Peptide XII had clearly different chromatographic pro­perties when compared with compound Xib and it was possible to prove that no tracesof the original sulfide were present in the sulfoxide prepared. Because of our inten­tion to use amino acid derivatives protected with the 2-nitrobenzenesulfenyl group

Collection czechostov. Chern. Commun. IVoL 43] [1978]

1544

IVa, R = MelVI>, R ~ H

Hcy(C,H,COOR)

VIla. R = Me1'1/1>_ R = H

Cys(Mc)

II

Cys(O) (C,I-I"COOR)

Va, R = MeVb. R = H

Hcy(O) (C,I-l,COOMc)

1'/11

Cys(O) (Me)

III

Lebl, Barth, Jogt :

VIa, R = MeVIlJ. R = H

Hcy(O,) (C,I-l,COOMc)

IX

Z-Cys(O) (C,I-l"COOMc)_(C"H, ,),NH

X

X-Cys(C,H,COOMc)-Pro-Leu-Gly-NH,

Xla, X~ZXII>, X = I-l

Cys(O) (C,H6COOMe)-Pro-Leu-Gly-NH,

XI/

Nps-Asn-Cys(O) (C,H,COOMe)-Pro-Lcu-Gly-NH,

X/II

X-Gln-Asn-Cys(O) (C,H6COOMe)-Prn-Lcu-Gly-NI-l,

XIVa, X ~ Nps; XIVI>.X~H

X-lIe-Gln-Asn-Cys(O) (C,H,COOR)-Pro-Leu-Gly-NI-I,

X Va, X = Nps, R = MeXVI>. X = H. R = I-I

Nps-Tyr(Eu' )-lIe-Gln-Asn-Cys(O) (C,H,COOI-I)-Pro-Leu-Gly-NH,

XVI

for the further building up of the peptide chain it was necessary to examine whetherthe sulfoxide group of the molecule would not be influenced when the prolectinggroup was split off. The stability of sulfoxide was proved by splitting off this group

Collection czecncelov. Chern. Commun. [Vol. 431 [1978]

Sulfoxides of Carba-Analogues of Oxytocin 1545

from 2-nitrobenzenesulfenylasparagine in mixture with sulfoxide Va. Peptide XIIwas prolonged by the 2,4,5-tricWorophenyl ester of 2-nitrobenzenesulfenylasparagine;this resulted in appearance of water soluble pentapeptide XIII. When the protectinggroup was split off hexapeptide XIVa was prepared using the same active esterof 2-nitrobenzenesulfenylglutamine. This peptide was also soluble in water and partof it was filtered through Arnberlite IR-4B. The protecting group was split off by HCIand the hydrochloride generated was transformed into the free peptide XIVb by filtra­tion through Amberlite IR-4B. Alternatively the oxidation-hydrolytic method/"was applied at this stage for the removal of the 2-nitrobenzenesulfenyl protectinggroup. This method based on the simultaneous action of sodium periodate andwater gave the free peptide in a high yield. The resulting peptide XIVb acylatedby the N-hydroxysuccinimide ester of 2-nitrobenzenesulfenyliwleucine gave a poorlywater soluble peptide XVa. The amino-protecting group was split off and methyl­ester was hydrolyzed by aqueous sodium hydroxide. Peptide XVb was then acylatedby N-hydroxysuccinimide ester of 2-nitrobenzenesulfenyl-O-tert-butyltyrosine. Theoctapeptide XVI obtained was converted into the active ester by treatment withbis(p-nitrophenyl) sulfite and after the amino-protecting group was split off cycliza­tion was performed in the usual manner'. Then, the protecting group on tyrosinewas removed by means of trifluoroacetic acid and the analogue I a thus preparedwas purified by counter-current distribution and gel filtration. In the counter-cur­rent distribution in the system 2-butanol-0'Oi% aqueous acetic acid, sulfoxide I a

had a different distribution coefficient than peptide Ie (K = 1'30 and 2'15, resp.)and it was therefore possible to exclude the contamination of analogue Ie by theproduct of sulfur oxidation if the final hormone analogue was purified by counter-cur­rent distribution. The chromatographic properties of sulfoxide Ia (synthesized orobtaiued by a direct oxidation) differ from those of sulfide Ie into which sulfoxide Iamay be converted by reduction.

A series of methods for sulfoxide reduction was published 2 ' - 31 but only a partof them may be used for our purposes"-'6. We tested the reduction of model com­pounds (III, Va) by means of 2-chloro-l,3,2-benzodioxaphospho]e37 (giving a mix­ture of products) and 2-mercaptoethanoI2 0

, which turned to be utilisable but dueto reaction conditions (50'C, 24 h) not the best one. Considering the reaction condi­tions the reduction by hydrogen bromide and acetone-? (a procedure described sometime ago but rarely used) proved to be best. The mild reaction conditions (20'C,5 min) are comparable only with dichloroborane reduction." (O'C, several min).It was verified that under the mentioned conditions the tyrosine residue was notdamaged and no cleavage of the molecule occurred even at reaction time longerby the magnitude of ten. The yields of the reduction were nearly theoretical whenionex chromatography was used for the product isolation. By the above-mentionedmethod sulfoxide Ia was reduced giving sulfide Ie with full biological activity; in thiscase the product was isolated by gel filtration.

Collection caecncslcv. Chern. cern-nun. [Vol. 43J 119781

1546 LebI,Barth, Jogt :

The biological activities of oxidized monocarba-analogues together with the activi­ties of some reference compounds are presented in Table IV. Compound I a had boththe activities tested, i.e. uterotonic in vitro'? and milk-ejectingr", lowered and dearlydissociated. The biological activities of a sample prepared by the oxidation of Iewere approximately same as those found for the sample prepared by the stepwisesynthesis. Significantly higher potencies were fonnd for sulfoxide Ie, they representedone half of the values estimated for the corresponding sulfide and this decrease wasproportional for both the activities. The oxidation of carba-analogue with main­tained amino group led to a decrease of potencies by several magnitudes of ten,justas it was found for the oxidation of deamino-l-carba-oxytocin to the sulfone stage.No inhibitory properties of the compound Ib were observed in the uterotonic assay(ratio Ibjoxytocin, 400: I).

It has been mentioned in many publications that the sulfur atoms of the oxytocinmolecule are not equivalent. Differences in biological potencies were found fordeamino-f-carba- and deamino-o-cnrba-oxytocinsf '<f just as in the case of mono­seleno analogues of deamino-oxytocin", Also the chiroptical properties of mono­carba-analogues were differenr':'. The great difference between the activities of I a

and Ie showed that non-equivalency of sulfur atoms was more evident when theywere oxidized instead of having been replaced by methylene or by a selenium atom.Concerning the biological activities of sulfoxide the general tendency was similarto selena-analogues where compounds of the l-seleno series were more active as com­pared with 6-seleno-analogues; in our case the analogues possessing sulfoxide groupin position I (i.e. analogues of the 6-carba series) had higher activities. Though we didnot verify it experimentally, we did not consider it provable that the differencein the activities of compounds Ia and Ie was caused by a different metabolic stability

TABLE IVBiological Activities of Oxytocin Analogues"

Analogue Isolated rat uterus

La 13·1Ib 0'05lc 455'4ld 0·26Ie I 899 b

If 929'Ig 734d

a Activities given in LV.fmg; b ref.4 1; c ref.4 2 ; d ref.4 8 .

Milk-ejection(rat)

91·0

0'03216'2

0'02604'3"456'5"142c

Collection Czechoslov. Chern. Commun. [Vol. 431 [19781

Sulfoxides of Carba-Analogues of Oxytocin 1547

of the analogues during their interaction with receptors or different eliminationrates from the receptor compartment. The availableinformation on the oxytocinreceptor (uterus) is not sufficient to support fully the correctness of the hypothesisaccording to which the agent intrudes "the receptor pocket" by a part of disulfidebond belonging to the sulfur atom in position 6 and that sulfoxide derived from thissulfur (i.e. sulfoxide of the l-carba series) cannot attain such a similar "receptorfit" due to spatial reasons. It is more plausible to cousider that the spatial position ofthe tripeptide side chain is iufluenced by a strongly polar group in position 6. Fromthe proposed secondary structuret" of oxytocin as well as from the measurements ofdiffusion rates through membranes's follows that the tripeptide side chain is orientedabove the cyclic part of molecule. A priori one cannot exclude the interaction of thesulfoxide in position 6 with this chain, mainly the effect on the hydrogen bond formedby the amino group of glycine and the carbonyl group of cysteine in position 6which concenquently forces the adoption of a less suitable conformation of thetripeptide side chain in its interaction with the receptor. Taking in account the modelof oxytocin proposed by American authors?" for the conformation of hormonein water, it is evident that the oxidized sulfur in position 6 influenced dramatically theabove mentioned hydrogen bond whereas the oxidized sulfur in position I waswithout effect. Sulfoxide in position I could affect the hydrogen bond between theamino-group of cysteine in position I and the carbonyl group of glycine which,however, is excluded in the case of deamino-analogue Ie. Therefore, the oxidationof this sulfur did not have a pronounced effect on the activity of deamino-ti-carba­-oxytocin. The significantly lower activity of sulfone Ib could be explained by highersteric requirements ofthe-S02- group or by higher probability of effecting a hydro­gen bond between the side-chain and the cyclic part of the molecule, in comparisonwith sulfoxide I a in which only one of the diastereoisomers could disturb this hydro­gen bond (ef.46). We cannot be sure that sulfoxideIa is exclusively ofone configurationalthough measurements of dichroic spectra of model sulfoxides/" show that theconfiguration of the sulfoxide sulfur changes quite simply and becomes thermodynami­cally more favorable.

The explanatiou of the extremely lowered activity of sulfoxide Id may be basedon the preferential formation ofa hydrogen bond between the sulfoxide group and theamino group of cystein in position I (wbich in oxytocin is engaged in a hydrogenbond with the glycine carbonyl) as a result of which not only the conformationof the cyclic part of the molecule is altered but also that of the tripeptide side chain.

EXPERIMENTAL

Samples for elemental analysis were dried for 24 h at room temperature and 1 Torr, Thin-layerchromatography (TLC) was carried out on silica gel plates (Silufol, Kavalier) in the solventsystems: 2-butanol-98% formic acid-water 75: 13-5: 11,5 (Sl), z-butanct- 25% ammonia-water

Collection Czechoslov. Chern. Commun. [Vol. 43] 1197BJ

1548 Lebl, Barth, Jest :

85: 7-5: 7'5 (82), l-butanol-acetic acid-water 4:J : 1 (83) and pyridine-Lbutancl-acetic acid­-water 10: 15: 3 : 6 (84). Paper chromatography was carried out on Whatmann 3MM paperin methanol-water-pyridine 85: 15 : 4 (85). Electrophoresis was performed in a wet chamberapparatus in 1M acetic acid (pH 2'4) and in pyridinium acetate buffer (pH 5-7) at a potentialdrop of 20 Vfernfor 60 min. Detection was by means of ninhydrin or by chlorination in the caseof ninhydrin-negative compounds. Samples for amino-acid analysis were hydrolyzed for 20 hat 105QC in 6M-HCI (in ampoules sealed at 1 Torr). The analyses were carried out Onan automaticanalyser (Development Workshops, Czechoslovak Academy of Sciences, Prague, type 6020).For evaporation we used rotatory evaporators (water pump, bath temperature 30-40°C).Mixtures containing dimethylformamide were evaporated at 1 Torr. Melting points were deter­mined on a Kofler block and values are uncorrected. For counter-current distribution the all­-glass Steady State Distribution Machine from Quickfit & Quartz, Ltd, Stone. Staffordshire,England. with independent shifting of both upper and lower phases, was used. In all cases thesolvent system was 2-butanol-0'05% aqueous acetic acid and the peptide material was locatedby the Folin-Ciocalteau reaction. For gel filtration we used the columns (100 X 1 em) of Bie-gel(Bio-Rad Laboratories) P-2 and P-4.

S-(y-Methoxycarbonylpropyl)cysteine Sulfoxide (Va)

a) To the solution of S-(y-methoxycarbonylpropyl)cysteine3 (IVa) (3'15 g) in water (180 ml)sodium periodate (4 g) was added and the mixture was stirred 2 h at room temperature. Afterfiltration. the mixture was cooled to O°Cand placed on a column of Dowex-Sf (H+ -cycle, 100 ml).The ion exchanger .was washed with water (O°C, negative test for iodate ions) and the productwas eluted with 10% pyridine (O°C). The eluate was evaporated and the residue was crystallizedfrom water and acetone; the yield was 3'05 g (90%) with the m.p. 139-143cC. The sample foranalysis was recrystallized in the same manner. m.p. 144-145°C; [«]55 _10'9° (c 0'2. water).For C,H15NOsS (237'3) calculated: 40'50% C, 6'37% H, 5'90% N; found: 40'67% C, 6'57% H,5-91% N.

b) To the solution of IVa (22 mg} in water (3'5 ml) potassium periodate (25 mg) was addedat 22°C. The samples were withdrawn after 5, 10, 30 and 60 min and the amount of sulfoxide(Va) in the reaction mixture was determined by means of paper electrophoresis. The conversionis a first-order reaction with a reaction constant equal to 1'1 . 10-3 s-1.

c) The suspension of IVa (220 mg) in a mixture of water (4'5 ml), methanol (2'5 ml) and 30%H20 2 (0'3 mI) was agitated 1 h at room temperature. The resulting solution contained 45%of sulfoxide Va and 55% of unreacted sulphide IVa. After further 24 h of standing at room tem­perature the product was precipitated by the addition of acetone; the yield was 190 mg of a com­pound, rn.p. 131-133°C, containing about 1% of sulfone VIa,

d) To the solution of IVa (220 mg} in water (25 mI) 100}.ll of Br2 was added. After mixing,the colour of the mixture immediately disappeared and pH value became lower. The solutionwas freeze-dried and the oily product which was obtained, contained the same amount of sulfideIVa and sulfoxide Va (TLC).

e) Compound IVa (220 mg) was suspended in chloroform (25 ml) and the solution obtainedafter addition of IOO}.lI of Br2 was evaporated 5 min later, the residue was dissolved in waterand freeze dried. The same mixture of compounds was obtained as in the previous attempt.

Collection caechoetov. Chern. Cornrnun. IVaI. 43] [19781

Sulfoxides of Carba-Analogues of Oxytocin 1549

Hydrochloride of SM(y-Methoxycarbonylpropyl)cysteine Sulfoxide (Va. HC!)

To the solution of sulfoxide Va (237 mg) in methanol (6 ml) 2M-HCI in ether (0'5 ml) was addedand the product was precipitated by the addition of ether (40 ml). Crystallisation from methanoland ether yielded 220 mg (81%) of the product with m.p. 126-128°C; [C(]fis 0° (c 0'2, water). ForC,H16CIN05S (273'7) calculated: 35·10% C, 5'89% H, 5'12% N; found: 34'89% C, 6·01% H5'15% N.

S-(y-Carboxypropyl)cysteine Sulfoxide (Vi

To the solution of methyl ester Va (237 mg} in water (2 ml) IM-NaOH (2 ml) was added. After1 h at room temperature the solution was cooled down to O"C and filtered through the columnof Dowex 50 (H +-form, 10 ml). The column was washed with water (O"C) and the productwas eluted with 10% pyridine. The eluate was taken to dryness, yield 200 rug (90%), m.p. 163 to165°C, RF 0'10 (SI), 0'00 (S2), 0'13 (S3), 0'14 (S4); El~~' 0'26. The sample for analysis was crystal­lized from methanol without change in the melting point; [C(]fi5 -4'5" (c 0'2, water). For C7H1J o

.N05S (223'2) calculated: 37'65% C, 5-87% H, 6'28% N; found: 37'41% C, 5'76% H, 6'12% N.

S-(y-Methoxycarbonylpropyl)cysteine Sulfone (VIa)

a) Compound IVa (220 mg) was oxidized 1 h at room temperature with performic acid, pre­pared from formic acid (4 ml) and 30% H 20z (0'3 ml). Lyofilisation afforded 250 mg (99~,J

of the product VIa, m.p. 158-159QC (decomp.). The sample for analysis was crystallized fromwater and acetone without change in m.p.: [C(]fis _6'0° (c 0'2, water). For CgH1SN06S (253'3)calculated: 37'94% C, 5·97% H, 5'53% N; found: 37·63% C, 5'71% H, 5'34% N,

b) When the reaction as described under a) above was carried out at _15°C a mixture of 25%sulfoxide Va and 75% sulfone Via was obtained.

c) To the solution of IVa (220 mg) in a mixture of water (1'5 ml), concentrated hydrochloricacid (0,2 ml) and methanol (Z'5 ml), 10% H 20Z (0'8 mI) was added. After 1 h at room tempera­ture methanol was evaporated and the mixture was freeze-dried. The yield was 240 mg of hydro­chloride VIa, m.p. 146-148°C, chromatographically identical with the compound prepared asunder a) above. •

S-(p-MethoxycarbonylethyI)homocysteine Sulfoxide (VIII)

Analogously to the preparation of the compound Va (oxidation with NaI04 ) sulfoxide VIiiwas prepared from VIla (0'5 g) in a 88% yield, m.p. 180-18ZoC, [0:]55 +13'0° (c 0'2, water).For C,H15NOsS (237'3) calculated: 40'50% C, 6'37% H, 5'90% N; found: 40'71% C, 6'41% H,5'95% N. RF values are given in Table 1.

S-(J}-Methoxycarbonylethyl)homocysteine Sulfone (IX)

Oxidation of VIla (0'5 g) with performic acid afforded sulfone IX. The yield was 0'49 g (90%),m.p. 224-225°C (decomp.), [0]5' +11'4° (c 0·2, water). For C,H15N06S (253-3) calculated:37'94% C, 5'97% H, 5'53% H; found: 38'Z5% C, 6·20% H, 5'47% N. RF values are given in Ta­blc 1.

Collection czechcalov. Chern. Commun. {Vol. 43] (1978]

1550

Reduction of Sulfoxides III and Va

Lebl, Barth, Jest :

a) To the suspension of Va (340 mg) in acetone (5 rot) 35% hydrogen bromide in acetic acid(2 rot) was added and after 5 min at room temperature the resultant solution was evaporated.After briefly drying (1 Torr) the residue was suspended in acetone (5 rot), the solvent was eva­porated and this operation was repeated once more. The remnant was dissolved in water (10 ml)and the solution was passed through Amberlit IR-4B (OH- -form, 25 ml) column. Evaporationof the eluent afforded 292 mg (92%) of the product, m.p. 200-207°C (ref. 3 gives m.p. 209 to212"C). Amino-acid analysis revealed 3% of Ivb but no sulfoxide Va.

h) Reduction of sulfoxides III and Va with 2-chloro-l,3,Z-henzo-dioxaphosphole was car­ried out according to ref.37; a mixture of unidentified products (TLC) was obtained.

c) Reduction of sulfoxides III and Va with 2-mercaptoethanol (according to ref,z°) giveschromatographically pure (TLC) sulfides II resp. IVa.

Dicyclohexylammonlum Salt of N-Benzyloxycarbonyl-S-(r-methoxycarbonylpropyl)cysteineSulfoxide (X)

a) To the solution of sulfoxide Va (1'5 g) in water (40 ml) sodium benzyloxycarbonyl thiosul­phate (2'5 g) was added and the pH was maintained at 7 by the addition of 2M-NaHC03 (21 ml)After 2 h no more NaHC03 is consumpted and the pH raises spontaneously. The mixture wasacidified with IM-HCI to pH 3, extracted with ether (4 X 20 mI), ethereal layers were combined,washed with water and dried. After evaporation, the remnant was dissolved in benzene, dicyclo­hexylamine (1'6 ml) was added. the solution was diluted by the addition of light petroleum,cooled to O°C and crystals which separated were collected, dried and crystallized from ethylacetate and light petroleum. The yield was 0'34 g (10%) of a product with m.p. 101-107°C.The sample for analysis was recrystallized in the same manner, m.p. 111-U5eC, [iX]55 -20'2°(c 0-2, dimethylformamide). For C2BH44N207S (552-7) calculated: 60-84% C, 8-02% H, 5-06% N;found: 60'56% C. 7·74% H, 5'39% N. The TLC comparison of the product with dicyclohexyl­ammonium salt of N-benzyloxycarbonyl-S-(r-methoxycarbonylpropyl)cysteine3 (the values in pa­renthesis): RF ; 0-65 and 0-48 (0-76 and 0-48) (SI), 0-16 and 0-63 (0-26 and 0-63) (S2), 0-58 and 0-35(0-74 and 0-35) (S3)_

b) To the solution of Va (385 mg) in water (l ml) 4M-NaOH (pH 8'5. 0'4 ml) was added and,under stirring and maintaining the pH at 8'5 (total 1'5 ml of 4M-NaOH). benzyloxycarbonylchloride (0'5 ml) was added. After 2 h the consumption of NaOH ceased. The mixture was dilutedby water (2 mI), extracted with ether, the aqueous layer was acidified to pH 3 with concentratedhydrochloric acid (0'5 ml) and the product was taken up in ethyl acetate. The organic layerwas washed with water, dried and evaporated; the yield was 487 rng (81%) of an oil with RF 0·65_(SI), 0-16 (S2), 0-58 (S3), 0-65 (S4); [aJI;' -30-3" (c 0-2, dimethylformamide). From a part of thisoily product the dicyclohexylammonium salt was prepared, identical with the One prepared asunder a) above.

The Splitting of the Protecting Groups in the Presence of Sulfoxide

a) Amino acid was liberated from its dicyclohexylammonium salt (X) (100 mg) by meansof 0·025M-H2S04 • The oil, obtained by usual working up of the mixture, was dissolved in aceticacid (0'1 ml) and 35% HEr in acetic acid (0'2 ml) was added. After 15 min at room temperaturethe product was precipitated by the addition of ether. TLC analysis revealed the presence of 5%of sulfide.

Ocllectlon Czechoslov. Chern. Commun. IVol. 43] [19781

Sulfoxides of Carba-Analogucs of Oxytocin 1551

b) To the solution of 2-nitrobenzenesulfenylasparagine (143 mg) and sulfoxide Va (120 mg)in methanol (20 ml) 2M-Hel in ether (0'80 rnl) was added. The mixture was evaporated to smallbulk and during trituration with ether it turned to solid state. Paper electrophoresis as well asquantitative amino-acid analysis revealed only asparagine and sulfoxide Va in the reaction mix­ture.

Sulfoxide of S-(y-Methoxycarbonylpropyl)cysteiny]-pro!yl.leucyl-glycine Amide (XII)

To the solution of the protected peptide Xla (4'5 g) in acetic acid (22'5 ml) 35% RBr in aceticacid (45 ml) was added. After 10 min at room temperature the mixture was diluted with ether(450 ml), the precipitate formed was triturated with ether, collected. dried and dissolved in water(100 ml). The aqueous solutions was passed through a column of Amberlite IR-4B (acetateform, 60 ml) and eluted with water. A part of the eluate was withdrawn for comparison with theproduct of the oxidation reaction. To the remaining eluate (270 ml) sodium periodate (2 g)was added and the mixture was stirred at room temperature for 2 h. The cooled (ocC) solutionwas transferred to a column of Dowex 50 (H + -cycle, 60 ml), the column was washed with water(Dec; negative reaction for iodate ions) and the product was eluted with 10% pyridine (OCC).The solution was taken to dryness, dissolved in a small amount of methanol, diluted with ben­zene and evaporated; this operation was repeated once more. After the last evaporation. the resi­due was dissolved in methanol. diluted with ether, and the oil which was obtained crystallizedon standing (contrary to the sample of sulfide where an attempt at crystallization failed). Theyield was 3'3 g (90%) of a product with m.p. 75-79"C; [(X]fi5 -46'5" (c 0'2. dimethylformamide).For C21H37Ns07S.2 H 20 (539'7) calculated: 46·74% C, 7'66% H, 12'97% N; found: 46'61% C.7·35% H, 12'99% N. TLC comparison with unoxidized sample X/b (the values are given in paren­thesis: RF 0·06 (0'12) (St), 0'03 (0'07) (S2), 0'06 (0'07) (S3), 0'29 (0'41) (S4)) proved that the pro­duet did not contain sulfide X/b.

SuIfoxide of 2-Nitrobenzenesu1fenyIaspar aginyI~S~(r ~methoxycarbonyIpropyl)cysteinyl-proly1­-leucyl-glyclne Amide (XIII)

To the solution of the tetrapeptide XII (3·2 g) in dimethylformamide (45 ml), Neethylpiperidine(0'4 mI) and 2,4.5-trichlorophcnyl ester of 2-nitrobenzenesulfcnylasparagine (3'6 g) were added.After standing at room temperature for 18 h a further base (0'6 ml) and active ester (l'8 g) wereadded. After 72 h. the mixture was evaporated. the residue was triturated with ether and crystalswhich formed were collected and washed with ether. The product is water soluble and thereforewater washings must be avoided. Precipitation from a mixture of methanol. dimethylformamideand ether yielded 3'2 g (63%) of a chromatographically pure product with m.p. 176-179"C;RF 0·27 (SI), 0'17 (S2), 0'24 (S3), 0'55 (S4) and 0'41 (CHCI3-methanol; 4: 1). The sample foranalysis was reprecipitated in the same way, m.p. 183-186"C; [«]55 -82'9" (c 0'2. dimethyl­formamide). For C31H46NsOllS2.H20 (788'9) calculated: 47'20% C, 6'13% H. 14'20% N;found: 47'39% C, 6'53% H, 14'29% N. Amino-acid analysis: Asp 1'01. Pro I-DO. Gly 1'02. Leu0'99, Cys(C3H6COOH) 0'50.

Sulfoxide of 2-NitrobenzenesulfenyJglutaminyl~asparaginyl~S~(r-methoxycarbonylpropyl)cys­

teinyl-prolyl-leucyl-glycine Amide (X/Va)

To the solution of protected pentapeptide XUI (3'1 g) in dimethylformamidc (40 ml) 2M-HeIin ether (2 ml) was added. After 4 min at room temperature the solution was diluted withether

Collection Czechoslov. Chern. ccmmun. [Vor. ~31 119701

1552 Lebl, Barth, Jost :

(200 ml), the precipitate was collected, washed with ether and dried. It was then dissolved in di­methylformamide (40 ml), the solution was alkalinized with Ncethylpiperidine (1'1 mI) and2,4,5-trichlorophenyl ester of 2-nitrobenzenesulfenylglutamine (2·5 g) was added. After 12 hat room temperature a second portion of Neethylpiperidine (0-4 rot) was added and 32 It laterthe mixture was taken to dryness and the residue was triturated with ether. Precipitation frommethanol and ether provided 4'1 g of a mixture of hexapeptide X/Va and Neethylpiperidinehydrochloride. A part of this mixture (0'5 g) was dissolved in water (20 rnI) and filtrated througha column of Amberlitc IR-4B (acetate form; 10 mI). The eluatewas evaporated, dissolved in ben­zene, taken to dryness and precipitated from a mixture of methanol, dimethylformamide andether. The yield of chromatographically and electrophoretically pure product was 0·44 g (88%),m.p. 11I-1I4"C. RF 0'24 (51), 0'21 (52),0'09 (53), 0'55 (54).E~:7 0'32, EY!X 0·60 (after splittingoff the protecting group). The total yield equals 100%. The sample for analysis was reprecipitatedfrom dimethylformamide and ether, m.p. 122-125"C; [a15s - 63-9" (c 0'2, dimethylformamide).For C36Hs4NI0013SZ.HzO (917'0) calculated: 47'15% C, 6'16% H, 15-27% N; found: 47'22%C,6'19% H, 14'98% N.

Sulfoxide of GlutaminyI-asparaginyl-S-(y-methoxycarbonylpropyl)cysteinyl-prolyI-leucyl­-glycine Amide (XIVb)

a) To the solution of hexapeptide X/Va (3'5 g of a preparation of 88% purity, i.e. 3·08 g of purecompound) 2M-Hel in ether (1'95 ml) was added. After 4 min at room temperature the solutionwas diluted with ether (300 ml), the precipitate obtained was triturated with ether and reprecipitat­ed from methanol with ether. After dissolving in water (100 ml) it was passed through a columnof Amberlite IR-4B (acetate form; 60 ml), the product was eluted with water, evaporated andtwice recrystallized from methanol and ether. The yield was 2'56 g (96%), m.p. 131-138"C;E~.iJO-47, Er.', 0'35, RF0-48 (S4)_ The sample for analysis was recrystallized in the same manner,mop. 135-142°C; [a]fi5 -55-2" (c 0'2, dimethylformamide). For C30HSIN901lS_HZO (763'9)calculated: 47'17% C, 6'99% H, 16'50% N; found: 46'87% C, 6'71% H, 16'24% N.

b) To the solution of hexapeptide XIVa (180 mg) in water (25 ml) NaI04 (90 mg) was added.After 4 h at room temperature (the colour of the mixture disappeared) the solution was passedthrough a column of Dowex-50 (H+-cycle; 4 ml), the ion exchanger was washed with waterand the product was eluted with 10% pyridine. The eluate was evaporated and the residue wasprecipitated from methanol and ether. The yield was 130 rug (87%) of a product of the same pro­perties as had the compound prepared as under a) above.

Sulfoxide of 2-Nitrobenzenesulfenylisoleucyl-gjutaminyl-asparaginyl-S-(y-methoxycarbonyl­propy1)cysteinyl-prolyl-Ieucyl~glycineAmide (XVa)

To the solution of XI Vb (2,5 g) in dimethylformamide (35 ml) Nehydroxysuccinlmide esterof z-nltrobenzenesulfenyllscleucine (2'3 g) and Ncethylpiperidine (250}JI and after 2 h 100 }JI)was added, After 70 h stirring at room temperature the mixture was evaporated, the residue wastriturated with ether, the crystalline portion was collected and washed with ether, ethyl acetate,water, ethyl acetate and ether. The crystallization from dimethylformamide, methanol and etheryielded 2·67 g (82%) of the product with m.p. 210-214"C; RF 0·24 (51), 0'20 (52), 0'12 (53),0-63 (S4)_ The sample for analysis was recrystallized from dimethylfcrmamlde and ether. m.p.217-219"C; [alii' -39'7" (c 0'4, dimethylformamide). For C42H6,Nll01452.2 H20 (1042)calculated: 4S-13% C, 6-63% H, 14'70% Nj found:4S-32% C, 6'34%H, 14-42% N.

Collection Czechoslov. Chern. Commun. iVaI_ 43] [197B}

Sulfoxides of Carba-Analogues of Oxytocin 1553

Sulfoxide of IsoleucY]MglutaminyI-asparaginyl-S-(y-carboxypropyl)cysteinyI-prolyI-leucy].-glycine Amide (XVb)

To the solution of heptapeptide XVa (2'0 g) in dimethylformamide (60 ml) 2M-Hel in ether(2'4 mI) was added. After 5 min at room temperature the mixture was diluted with ether (300 ml),the precipitate was collected. washed with ether and dried; Rp 0'05 (81), 0-07 (82), 0'08 (83),0'41 (84), E~5ro·42. The compound was dissolved in water (50 ml), the solution was alkalinizedwith IM-NaOH (6 ml, pH 12) and after 25 min the pH was brought to the value of 7 (5'7 mlof IM-Hel). The solution was cooled (DOC), transferred to a column of Dowex-50 (H+-cycle,60 rot), the column was washed with water (O°C) and the product was eluted with 10% pyridine.The eluate was evaporated, dissolved in a mixture of methanol and benzene and evaporated,The precipitation from dimethylformamide, methanol and ether yielded ]'3 g (77%) of theproduct with m.p. 181-185"C; RF 0·05 rsn, 0'01 (S2), 0·08 (S3),0'28 (S4), E1/!i 0'12, Er.~ 0'43.The sample for analysis was reprecipitated from dimethylformamide and ether, m.p. 186 to189°C, [fZ]fl -52'4° (c 0'2, dimethylformamide). For C3sH60N10012S.2·5 H20 (890'0) cal­culated: 47'23% C, 7'36% H, 15'73% N; found: 47'25% C, 6'98% H, 15'45% N. Amino-acidanalysis: Asp 0'99, Glu 0'97, Pro 1-02, Gly 1'01, IIe 0'99, Leu 1'01, Cys(C,H,COOH) 0'56.

Sulfoxide of 2-Nitrobenzenesulfenyl-O-tert-butyltyrosyl-isoleucyl-glutaminYI-asparaginyl­-S~(y-carboxypropyl)cysteinyl-prolyl-Ieucyl-glycineAmide (XVI)

To the solution of free heptapeptide XVb (1 g) in dlmethylformamide (50 ml), Neethylpiperidine(0'25 mI) and N-hydroxysuccinimide ester of 2-nitrobenzenesulfenyl-0-tert-butyItyrosine(1'3 g) was added. After 10 h more base (0'3 mI) was added and after additional 28 h the solu­tion was evaporated and the residue Was triturated with light petroleum and ether. The crystal­line portion was collected and washed with ethyl acetate, water, ethyl acetate and ether. The yieldwas 1'3 g (91%), m.p. 202-204"C, RF 0'47 (SI), 0'13 (S2), 0·73 (S3), 0'70 (S4). The sample foranalysis was crystallized from dimethylformamide and ether without a change in the meltingpoint; [fZ]i,S +1'0° {c 0'2, dimethylformamide). For C4sHsoN12 016S2.2 H20 (1253) calculated:51'74% C, 6'75% H, l3'41% N; found: 51'52% C, 6'55% H, 13'40% N. Amino-acid analysis:Asp 0'99, Glu 0'97, Pro 1'00, Gly 1'01, IIe 0'98, Leu 1'01, Tyr 0'98, Cys(C,H6COOH) 0'53.

Sulfoxide of Tyrosyl-isoleucyl-glutaminyI-asparaginy1~S-(y-carb oxypropyljcystelnyI-prol yl­-leucyl-glycine Amide Lactam (Ia)

a) To the solution of protected octapeptide XVI (500 mg) in dimethylformamide (15 ml) andpyridine (15 ml) was, under nitrogen, bisfp-nitrophenyljsulfite (1 g) added and after 6 h at roomtemperature a further sulfite (1 g). After 12 h another 0'5 g portion of sulfite was added and 5 hlater the mixture was evaporated, the residue was triturated with ether, collected, dried and dis­solved in dimethylfcrmamide (9 ml). To the solution 2M-HCIin ether (1 ml) was added and after6 min at room temperature the mixture was diluted with ether (300 ml), the precipitate was col­lected, washed with ether and dissolved in dimethylformnmide (20 mf). This solution was added,at a rate of 5 mljh, to a mixture of pyridine (320 ml) and N-ethylpiperidine (I50 J1.1) under heating(50°C) and nitrogen bubbling. The mixture was stirred 2 h at 50"C, then 14 h at room temperature,taken to drynessand the residue was triturated with ether. The oily product was dissolved in tri­fluoroacetic acid (15 mI) and after 50 min at room temperature the mixture was diluted withtoluene (15 ml) and evaporated. The residue was triturated with ether and the solid compoundwas dissolved in 25 ml of the upper phase of the solvent system 2-butanol-0·05% aqueous aceticacid, the filtered solution was placed in the second tube of the counter-current distributionmachine and 150 transfers of the upper phase and 60 transfers of the lower phase were carried

Collection czechoetov. Chern. Commun. [Vol. 43J [1978]

1554 LebI, Barth; Jost :

out. Three peaks with K = 0'14,0'60 and 1'30 were found. The first peak contained a ninhydrin­positive compound, the second one contained only a very small amount of peptide materialand was not analysed. The third peak with K= 1'30 (tubes No 49-73) was concentratedto a small volume and Iyofilized. The yield was 130 rug (31%). A part of this product (60 mg)was dissolved in 3M acetic acid (2 ml) and transferred to a column of Bie-gel P-4. Lyofilisationof a peak of biologically active material (localised by OD 2 8 0) yielded qri-Z'mg of the productidentical in all properties with the product obtained by oxidation. The sample for analysis wasprecipitated from methanol and ether, [~]fi5 _50'0° (c 0'12, water). For C44H67NllOt3S ..3 H zO.CzH40Z (J ]04) calculated: 50'03% C, 7-02% H, 13-95% N; found: 49-83% C, 6'70% H,13'82% N. Amino-acid analysis: Asp 1'00, Glu 0'99, Pro 1'02, Gly J '00, IIe 0'98, Leu 1'02, Tyr0'97, Cys(C3H.COOH) 0-47.

b) To the solution of deamino-f-carba-oxytocirr[ (Ie) (15 mg) in water (0'5 ml)sodium perle­date (6'4 mg) was added. After 2 h at room temperature the mixture was diluted with 3r.1 aceticacid (1 ml) and transferred to a column of Bie-gel P-2 (100 X I ern) in 3M acetic acid. Theproduct was localized by OD Z80 and Iyofilisation of the peak afforded 9·5 mg of the product.RF value are given in Table III.

c) The solution of deamlno-J-carba-oxytocin'' (Ie) (15 mg} in methanol (0'2 ml) was leftaside for 6 months at room temperature. TLC analysis revealed 30% of sulfoxide Ia.

Sulfone of Deamino-Iecarba-oxytocin (lb)

Deamino-l-carbu-oxytocin'' (Ie) (15 mg) was dissolved in a mixture (0'15 ml) of formic acid(4 ml) and 30% HzOz (0'3 ml), 30 min after the preparation of this mixture. After standingfor 1 h at room temperature, the solution was freeze-dried, the lyofilisate was dissolved in water(0'5 ml) and again freeze-dried. After dissolving in 3M acetic acid the solution was transferredto a colu~n of Bio-gel P-2; the yield was 11 mg. The sample for analysis was precipitated frommethanol and ether,[a]ft _65'5" (c 0'1, water). For C44H67N11014S.2 H zO.CzH40Z (1102)calculated: 50'13% C, 6'86% H, 13'98% N; found: 50'23% C, 6-57% H, 14'12% N. Amino-acidanalysis: Asp 1'02, GIn 1'00, Pro 1-03, Gly 1-01,!Ie 0'98, Leu 1-02, Tyr 0-97, Cys(Oz)(C3H._.CaOH) 0'95 (for calculation the constant for Cys(C3H6COOH) was used); no trace of Cys•.(C3H.COOH) was found.

Sulfoxide of Deamino-fi-carba-oxytocin (Ie)

To the solution of deamino-ri-carba-oxytocin" (If) (3'4 mg) in a mixture of water (200 Ill) andmethanol (20 JlI) a solution of sodium periodate (l'5 mg) in water (10 Ill) was added. After2 h standing at room temperature the mixture was worked up as described above; yield was2'1 mg. TLC analysis proved that the product contained no sulfide If, cf. Table III. Amino-acidanalysis: Asp 1'06, Glu l'OO,Pro 1'00, Gly 1'00, lie 0-97, Leu 1-04, Tyr 0-95, Hcy(C zH 4COOH)0-51.

Sulfoxide of t-Carba-oxytocln (Id)

To the solution of f-carba-oxytocin't'' (Ig) (5 rng} in a mixture of water (300 ul) and methanol(20 J.1I) a solution of sodiumperiodate (2,4 mg) in water (10 Ill) was added. After 2 h standing.at room temperature the mixture was worked up as described for Ia above. The yield was 2-5 mg,Rp values are given inTable III. Amino-acid analysis: Asp 1,00, Glu 0-98, Pro 1'02, Gly ·1'02,lie 0'98, Leu ]'00, Tyr 0'98, Cyth 0'54.

Collection Ozechcatcv. Chern. Commun. IVai. 431 [19781;

Sulfoxides of Carba-Analogues of Oxytocin

Deamino-J -carba-oxytocin (Ie)

1555

To the suspension of sulfoxide Ia (Z mg) in acetone (ZOO Ill) 35% HBr in acetic acid (100 Jl1)was added. The obtained solution was after 12 min standing at room temperature evaporated,the residue was suspended in acetone (2 ml ) and evaporated again. The remnant was trituratedwith ether, dried, dissolved in 3Macetic acid and transferred to a column of Bie-gel pw2 in 3Macetic acid. The procedure yielded I'Z mg of a compound chromatographically identical withdeernlno-t-carba-oxytocin, having 1790 LV./mg when tested on the isolated rat uterus.

Pharmacological Methods

The assay on the isolated rat uterus was carried out on strips of uterus of adult Wistar strainrats, under the influence of oestrogens. The strips were placed into the medium3 9 which wasbubbled through with 95% 02 - 5% CO2 at 30°C. Isometric contractions were recorded usinga magnetoelectric transducerd"; oxytocin was used as a standard. The inhibitory effect of analogueIb was measured by the shift of the dose-response curve. For the determination of the milk-eject­Ing activity, lactating female rats (5-14 days after delivery) of the same strain were used. Theassay was carried out as described in ref.4 0 •

Our thanks are due to Dr J. Smolikatxi for the interpretation of IR spectra, to Dr J. Krupickafor the polarographic determination and to Dr I. Frti for measuring and interpretation ofCts-speetra.We would like to thank Mrs H. Koudiood and Mrs J. Kelleroui for carrying out the pharmaco­logical tests, to Mrs H. Farkasota for amino-acid analyses and to Mrs Z. Ledoinotxi for measuringthe optical rotation. The elemental analyses were carried out in the analytical deportment of thisInstitute (head Dr J. Horacek).

REFERENCES

I. Toennis G., Callan T. P.: J. BioI. Chern. 129, 481 (1939).2. Tentatioe Rules Oil Biochemical Nomenclature. Biochemistry 6, 362 (1967); Biochem. J. 126,

773 (1972).3. .rosr K.: This Journai 36, 218 (1971).4. rest K., Sorm F.: This Journai 36, 234 (1971).5. Yamanaka T., Hase S., Sakakibara S., Schwartz 1. L., Dubois B. M., Walter R.: Mol.

Pharmacol. 6, 474 (1970).6. Walter R., Gordon W., Schwartz 1. L., QuadrifogIio F., Urry D. W.: Peptides 1968. Proc.

IXtlz Europ, Pept, Symp., Orsay 1968 (E. Bricas, Ed.}, p. 50. North-Holland, Amsterdam1968.

7. Barth T.: Unpublished results.8. Dixon H. B. F.: Biochim. Biophys. Acta 19, 392 (1956).9. Dedman M. L., Farmer T. H., Morris C. J.O. R.: Biochem. J. 78, 348 (1961).

10. Hofmann K., Rosenthaler J., Weiis R. D., Yajima H.: J. Amer. Chern. Soc. 86, 4991 (1964).11. Medzihradszky J.: Pep/ides 1976. XIVtlz Europ. Pept, Symp.• Wepion 1976 (A. Loffet, Ed.),

p. 401. Universite de Bruxelles, Bruxelles 1976.iz. Riniker B., Neher R, Maier R., Kahnt F. W., Byfield P. G. H., Gudmundsson T. V., Galante

L., Maclntyre 1.: Heiv. Chim, Acta 51,1738 (1968).13. Rasmussen H.: Science 128, 1347 (1958).14. Sandrin E., Boissonnas R. A.: Helv. Chim. Acta 47, 1294 (1964).15. Morley J. S., Tracy H. J., Gregory R. Q.: Nature (London) 207,1356 (1965).

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